![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Biochemistry, University of California-Riverside, Riverside, CA 92521 and * Department of Pathology, School of Medicine, University of California-Los Angeles, Los Angeles, CA 90024
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
A number of putative molecular chaperones seem to play essential roles in the correct folding, assembly and glycosylation of membrane and secreted proteins in the endoplasmic reticulum. We have shown that life span-extending dietary energy restriction significantly and specifically reduces GRP78 mRNA and protein by 50-75% in mice. Here, 5-mo-old female C3B10RF1 mice were given free access to food after being fed 50% less dietary energy since weaning. Hepatic GRP78 mRNA increased linearly, reaching the same level after 2 wk as was found in the liver of 20-mo-old mice with free access to food. This increase took place with no change in body weight. The mRNA levels of endoplasmic reticulum, cytosolic and mitochondrial chaperones were determined in young (7-mo-old) and old (21- or 28-mo-old) female C3B10RF1 mice. Each age group was either 50% energy restricted or was fed approximately 10% less energy than consumed by mice given free access to food. In young and old energy-restricted mice, hepatic expression of the endoplasmic reticulum chaperones ERp57 (37%), GRP170 (51%), ERp72 (43%), calreticulin (54%) and calnexin (23%) was significantly and specifically reduced. The GRP78, GRP94, GRP170, ERp57 and calnexin mRNA response to diet occurred reproducibly only in liver, and not in adipose, brain, heart, kidney, lung, muscle or small intestine. The mRNA for GRP75, a mitochondrial chaperone, HSC70, a cytoplasmic chaperone, protein disulfide isomerase, an endoplasmic reticulum chaperone, and C/EBP
, a transcription factor, was not regulated. Hepatic C/EBP
was 15% higher in old energy-restricted mice. Thus the expression of nearly all endoplasmic reticulum chaperones responded rapidly and specifically to dietary energy in mice.
INTRODUCTION
A number of molecular chaperones interact with nascent proteins in the endoplasmic reticulum to assist in their biosynthesis, processing and folding (Gething and Sambrook 1992
, Hammond and Helenius 1995). The glucose regulated proteins (GRP)4 are a family of stress-induced molecular chaperones (Buchner 1996, Little et al. 1994). Three members of this family reside in the endoplasmic reticulum: GRP78 (also called BiP; Haas and Wabl 1983), GRP94 (also called endoplasmin, ERp99, gp96, hsp100, hsp108; Gething and Sambrook 1992, Hendrick and Hartl 1993, Lee 1994), and GRP170. They transiently bind to a wide repertoire of proteins traversing the endoplasmic reticulum and facilitate their correct folding, glycosylation, assembly and turnover (Buchner 1996, Lin et al. 1993, Little et al. 1994, Mazzarella et al. 1994, Otsu et al. 1995). Expression of these genes is induced in cultured cells by agents that interfere with the normal glycosylation, folding or assembly of proteins in the endoplasmic reticulum (Lee 1994). The GRP78 protein shares 60% amino acid identity with members of the HSP70 protein family, and GRP94 shares approximately 50% amino acid sequence identity with HSP90 (Lee 1994). The glycoprotein GRP170 is structurally related to both HSP110 and HSP70 (Key et al. 1996). Antibodies to GRP170 coprecipitate GRP78 and GRP94, indicating these proteins associate in the endoplasmic reticulum (Lin et al. 1993). Another family member, GRP75 (also called PBP74, mtHSP70, mortalin and CSA; reviewed in Webster et al. 1994), is located in the mitochondria, where it probably acts as a molecular chaperone (Mizzen et al. 1989).
There are other chaperones in the endoplasmic reticulum. Three of these, protein disulfide isomerase (PDI), ERp57 (also called GRP58 and ERp61; Mazzarella et al. 1994) and ERp72 (also called CaBP2; Van et al. 1993), share a repeated amino acid sequence (thioredoxin domains) at their active sites (Lundstrom-Ljung et al. 1995, Mazzarella et al. 1990). Protein disulfide isomerase is an abundant protein that is loosely associated with the luminal surface of the endoplasmic reticulum membrane (Freedman 1984). It is responsible for isomerization of protein disulfide bonds during or shortly after synthesis to yield proteins with native disulfide bonds. ERp57 and ERp72 possess the thiol-dependent reductase activity of PDI (Lundstrom-Ljung et al. 1995, Mazzarella et al. 1990, Van et al. 1993) and cysteine protease activity (Hirano et al. 1995, Otsu et al. 1995, Srivastava et al. 1993, Stafford and Bonifacino 1991, Urade et al. 1992). ERp72 has been detected in complexes with denatured or incorrectly folded proteins and other chaperones, including GRP78 and GRP94 (Feng et al. 1995 and 1996, Kuznetsov et al. 1994). These results suggest that ERp72 functions in association with other chaperones to help mediate correct protein folding in the endoplasmic reticulum. Protein disulfide isomerase, ERp72 and ERp57 are induced by agents and treatments that disrupt protein processing in the endoplasmic reticulum, such as calcium ionophore (Dorner et al. 1990, Lee 1981, Van et al. 1993).
ERp57 and three other endoplasmic reticulum proteins, calnexin, calreticulin and 5
-diphosphate (UDP)-glucose:glycoprotein glucosyltransferase, are thought to mediate a glycoprotein-specific quality control cycle (Hammond and Helenius 1995, Helenius 1994, Oliver et al. 1997). This cycle ensures that only correctly folded and assembled proteins exit the endoplasmic reticulum to appear in the later compartments of the secretory pathway. Calnexin is an ubiquitous, Ca2+ binding, integral endoplasmic reticulum membrane protein that functions as a molecular chaperone. Its active site projects into the lumen of the endoplasmic reticulum. Calreticulin is an endoplasmic reticulum chaperone with sequence similarity to the endoplasmic reticulum luminal domain of calnexin (David et al. 1993). Along with GRP78, GRP94, ERp72 and PDI, calreticulin binds selectively to denatured proteins in a Ca2+ and ATP reversible fashion (Nigam et al. 1994).
Energy restriction (ER) delays most age-related physiologic changes, is the only method known for extending life span in homeothermic vertebrates, and is the most effective means known for reducing cancer incidence and increasing the mean age of onset of age-related diseases and tumors (Weindruch and Walford 1982 and 1988). We have previously shown that the mRNAs for hepatic GRP78 and GRP94 are significantly and consistently reduced in mice subjected to long-term ER (Spindler et al. 1990, Tillman et al. 1996a). Additionally, we have shown that ER produces a decrease in GRP78 protein that is equivalent to the decrease in mRNA (Tillman et al. 1996a). The reduction in GRP78 and GRP94 mRNA is proportional to the degree of ER (Spindler et al. 1990). Mice fed 50 and 20% of the energy consumed by mice with free access to food had progressively lower expression of hepatic GRP78. Recently, others have reported that GRP78 mRNA is reduced by ER in rats subjected to a methodologically and nutritionally different ER regimen, indicating the generality and robustness of the response (Heydari et al. 1995). Because specific changes in GRP78 levels can alter both the rate and extent of specific protein secretion (Dorner et al. 1988 and 1992), the changes in endoplasmic reticulum chaperone expression in ER mice may contribute to the extension of life and health spans by ER. Here we report the effects of 50% ER on the expression of endoplasmic reticulum, cytoplasmic and mitochondrial chaperones in eight tissues of mice.
MATERIALS AND METHODS
). Male C57BL10.RIII/Sn and female C3H.Sw/Sn mice obtained from Jackson Laboratories (Bar Harbor, ME) were bred and maintained at our animal facility. Mice were maintained at 20-24°C and 50-60% humidity with lights on from 0600 to 1800 h. Mice had free access to water. Sentinel mice were kept in the same room as the experimental mice, and serum samples were screened every 6 mo for titers against 11 common pathogens. No positive titers were found during these studies. Animal use protocols were approved by the appropriate animal use committees of the University of California, Riverside and the University of California, Los Angeles.
Diets.
Mice were weaned at 28 d, housed individually and subjected to one of the three diet regimens described below. The composition of the defined diets used in these studies has been described in detail (Spindler et al. 1990). Diets are defined and formulated so that dietary groups receive approximately equal amounts of protein, corn oil, minerals and vitamins per gram of body weight. The mice were fed and maintained as described previously (Spindler et al. 1990). Mice with free access to food (FA) consumed approximately 451 kJ/wk. Control mice consumed the control diet in amounts that provided approximately 406 kJ/wk. The 50% ER mice ingested restricted diet in amounts providing approximately 225 kJ/wk. The reduction in dietary energy was achieved by a reduction in the amount of carbohydrates consumed. The 50% ER mice were fed on Monday, Wednesday and Friday. On Friday the mice received 2.6 times the amount fed at the other feedings. The control mice were fed daily, except weekends. On Friday they were given a 3-d allotment of food. Feeding was between 0900 and 1100 h. All food was routinely consumed before the next feeding. The ER, control and FA mice weighed 22.4 ± 1.5, 40.6 ± 7.4 and 45.6 ± 9.4 g, respectively. For the studies shown in Figure 1, ER mice of 5 mo of age were divided into groups. RNA was prepared from the liver of one group of five mice. Ten other ER mice were given free access to food. After 1 wk, RNA was prepared from the livers of five of these mice. After a total of 2 wk of the FA regimen, RNA was prepared from the livers of the remaining five mice. RNA was also prepared from the livers of eight mice of 80 wk of age that had been either ER or FA since weaning. The mRNA for GRP78 was determined. Weights were determined at the time of use. For the studies of hepatic chaperone expression shown in Table 1, young (7 mo) and old (28 mo) ER and control mice were used. For the studies of gene expression in adipose, brain, heart, kidney, liver, lung, muscle or small intestine, 21-mo-old ER and control mice were utilized (n = 8). Food was removed from the cages 12 h before the mice were used.
|
Table 1.
The hepatic level of Calnexin, Calreticulin, C/EBP |
). The RNA were analyzed using Northern blots to verify their integrity. Dot blots were used to quantify mRNA levels (Spindler et al. 1990, Tillman et al. 1996a). Specific mRNA levels were normalized to the level of total mRNA present in each sample using hybridization with radiolabeled oligo-dT (Pharmacia Inc., Piscataway, NJ) and/or S-II cDNA as previously described (Hirashima et al. 1988, Tillman et al. 1996a). The murine ERp72 2.5-kb cDNA was excised with BamHI from pcD72-1 (Mazzarella et al. 1990). The 1235-bp murine GRP75 coding fragment was excised with HindIII from pG7z-PBP1.8 (Domanico et al. 1993). A 1370-bp murine calnexin coding fragment was produced by Pstl digestion of pRc/CMV (Schreiber et al. 1994). A 664-bp coding fragment of rat calreticulin (nucleotides 148-812) was produced by PCR from GT10.U1 (Smith and Koch 1989). The entire 2.4-kb cDNA of murine PDI was excised from pGEM59.4 with SacI and BamHI (Mazzarella et al. 1990). A 1-kb coding fragment of hamster GRP170 cDNA was excised with EcoRI and XhoI from pCRtmII (Lin et al. 1993). The 800-bp cDNA fragment of mouse C/EBP was excised with PstI from p+3mC/EBP (Christy et al. 1991). The entire coding fragment of C/EBP was used as a probe (Cao et al. 1991). The 1.9-kb cDNA of murine ERp57 was excised with HindIII and SstI from pERp61 (Mazzarella et al. 1994). The 1-kb cDNA of murine HSC70 was excised with PstI from phsc1.5 (Giebel et al. 1988). The fragments were isolated by agarose gel electrophoresis and radioactively labeled using a T7QuickPrime Kit (Pharmacia) according to the manufacturer's instructions.
Statistical analysis.
Values are expressed as means ± SD. The level of significance chosen was P < 0.05. To test for significance of the effects of age and diet on gene expression (Table 1), two-way ANOVA was used. No interactions between age and diet were observed. In Table 1, the significance of the differences between the four groups was tested by Tukey's pairwise comparisons. The effects of ER on gene expression in the different tissues were assessed using Student's unpaired t test. The responses of weight and GRP78 mRNA levels to the shift from ER to feeding FA (Fig. 1) were analyzed using Tukey's pairwise comparisons. Statistical analyses were performed with MINITAB Statistical Software, Standard Version, 1992 (Minitab Inc., State College, PA).
RESULTS
). To determine how many tissues respond in this fashion, GRP78 mRNA levels were measured in adipose, brain, heart, kidney, liver, lung, muscle and small intestine of ER and control mice. Hepatic GRP78 mRNA in these ER mice (1.07 ± 0.45 phosphorimager units) was ~35% of that found in control mice (3.05 ± 0.64 phosphorimager units; P < 0.001). However, GRP78 mRNA levels were not different in any of the other tissues (data not shown), indicating that this regulation is highly tissue specific.
, Spindler et al. 1990). Energy restriction produced no change in the ratio of polyadenylated RNA (mRNA) to ribosomal RNA in any of the tissues (data not shown; Mote et al. 1991b, Spindler et al. 1991).
, Nigam et al. 1994). Furthermore, this association has been shown to occur in vivo (Kuznetsov et al. 1994). ERp72 regulation was similar to that of ERp57 and the GRP (Table 1). ERp72 hepatic mRNA was significantly reduced approximately 43% in ER mice of both age groups (P < 0.001). The level of the mRNA increased 25% in the liver of the older mice (P = 0.001).
, Mizzen et al. 1989). It has 49 and 46% amino acid sequence homology with GRP78 and HSC70 (Webster et al. 1994). It is constitutively expressed, but its expression is induced by stresses that induce the endoplasmic reticulum GRP. These stresses include glucose deprivation and exposure to either calcium ionophore or 2-deoxyglucose (Domanico et al. 1993). Despite these similarities to the endoplasmic reticulum members of the GRP family, the mRNA for GRP75 was not differentially expressed in ER compared with control mice (Table 1, P = 0.436). Expression of GRP75 was not affected by age (P = 0.794).
Table 2.
Summary of the effect of 50% energy restriction on the hepatic chaperone mRNA expression in mice
). The mRNA for HSC70, the constitutively expressed homologue of HSP70, was not affected by ER (P = 0.457) but was significantly higher in older mice (Table 1, 31%, P < 0.003). Thus, even though both HSC70 and GRP75 are closely related to GRP78, they are not located in the endoplasmic reticulum (Table 2), and they are not regulated by ER.
mRNA in the liver of ER mice (Table 1, P = 0.023). There was no difference in C/EBP expression between control and ER mice (Table 1, P = 0.803).
DISCUSSION
).
to a lesser extent PDI are regulated in cultured cells by calcium ionophores and other agents that produce malfolded or incorrectly glycosylated proteins in the endoplasmic reticulum. However, the response to ER is restricted to a subset of these genes. The mRNA for the mitochondrially localized GRP75, which is coded for by a nuclear gene, and the mRNA for PDI, a luminal endoplasmic reticulum chaperone, were not regulated by ER. Thus, the genetic elements involved in the stress-related transcriptional response may not be involved in regulation by diet. We are presently conducting studies to identify the cis elements responsible for ER regulation of these genes. Identification of the DNA or RNA sequence element responsible for ER regulation should help explain the third novel result of these studies, the tissue specificity of the response.
is important in the differentiation of hepatocytes and adipocytes and in the establishment and maintenance of energy homeostasis (Wang et al. 1995). C/EBP is abundant in the liver. Its mRNA is 80% lower in the liver of rats with elevated blood insulin concentrations and higher in rats with lower blood insulin concentrations (Bosch et al. 1995). The 15% increase in C/EBP in ER mice may be a response to reduced blood insulin concentrations. The increase in C/EBP could result in subtle changes in gene expression important in the physiological response to ER.
, Koizumi et al. 1989, Masoro et al. 1992). Because of their unique role in serum glucose regulation, hepatocytes are among the very few cell types in which intracellular glucose concentrations tend to equilibrate with blood glucose concentrations (Craik and Elliott 1979, Gould et al. 1991). Thus, responsiveness to intracellular glucose levels could explain the liver specificity. We cannot at present distinguish among these possibilities.
and 1992, Lodish and Kong 1990). For example, overexpression of GRP78 by two- to threefold slows the rate of secretion of specific proteins. A 50-75% reduction in GRP78 levels improves secretion of other proteins. Thus negative regulation of many endoplasmic reticulum chaperones by ER may result in changes in the secretion efficiency of serum proteins. We have preliminary evidence that ER enhances the rate and/or extent of secretion of many serum proteins from hepatocytes. We speculate that this may lower the level of glycated protein in the serum by increasing the output and turnover of serum proteins. This change in secretion may be responsible for part of the reduction in glycated serum proteins and the reduced renal, vascular and other damage found in ER mice (Cefalu et al. 1995, Masoro et al. 1989, Sell et al. 1996). This kind of damage is associated with both diabetes and aging.
ACKNOWLEDGMENTS
cDNA, Steven McKnight (Tularik, Inc., South San Francisco, CA) for C/EBP cDNA, John Subjeck (Roswell Park Cancer Institute, Buffalo, NY) for GRP170 cDNA, Michael Smith (MRC Laboratory of Molecular Biology, Cambridge, England) for calreticulin cDNA and Larry Tjoelker (ICOS Corporation, Bothell, WA) for calnexin cDNA.
FOOTNOTES
and C/EBP, CAAT/enhancer binding proteins and ; ER, dietary energy restriction; ERp57 and ERp72, endoplasmic reticulum proteins 57 and 72; FA, free access to food; GRP, glucose-regulated protein(s); GRP75, GRP78, GRP94 and GRP170, glucose regulated protein 75, 78, 94 and 170; HSC70, heat shock cognate 70; HSP, heat shock protein; PDI, protein disulfide isomerase.
Manuscript received 13 March 1997. Initial reviews completed 14 April 1997. Revision accepted 13 May 1997.
LITERATURE CITED
identification of its secretory form and inducible expression by the oncogenic transformation.
Eur. J. Biochem.
1995;
234:336-342
[Medline]
results of a 17 year follow-up.
Br. Med. J.
1996;
313:775-779This article has been cited by other articles:
|
|
 |
|
  M. Leroch, H. E. Neuhaus, S. Kirchberger, S. Zimmermann, M. Melzer, J. Gerhold, and J. Tjaden Identification of a Novel Adenine Nucleotide Transporter in the Endoplasmic Reticulum of Arabidopsis PLANT CELL, February 1, 2008; 20(2): 438 - 451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ozawa, M. Miyazaki, M. Matsuhisa, K. Takano, Y. Nakatani, M. Hatazaki, T. Tamatani, K. Yamagata, J.-i. Miyagawa, Y. Kitao, et al. The Endoplasmic Reticulum Chaperone Improves Insulin Resistance in Type 2 Diabetes Diabetes, March 1, 2005; 54(3): 657 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Dhahbi, H.-J. Kim, P. L. Mote, R. J. Beaver, and S. R. Spindler Temporal linkage between the phenotypic and genomic responses to caloric restriction PNAS, April 13, 2004; 101(15): 5524 - 5529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zhang, G. Wu, C. G. Tate, A. Lookene, and G. Olivecrona Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21) J. Biol. Chem., August 1, 2003; 278(31): 29344 - 29351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Dhahbi, S. X. Cao, P. L. Mote, B. C. Rowley, J. E. Wingo, and S. R. Spindler Postprandial Induction of Chaperone Gene Expression Is Rapid in Mice J. Nutr., January 1, 2002; 132(1): 31 - 37. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. X. Cao, J. M. Dhahbi, P. L. Mote, and S. R. Spindler Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice PNAS, September 4, 2001; (2001) 191313598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Hansen Introduction J. Nutr., March 1, 2001; 131(3): 900S - 902. [Abstract] [Full Text]
|
|
|
|
|
|
R. Weindruch, T. Kayo, C.-K. Lee, and T. A. Prolla Microarray Profiling of Gene Expression in Aging and Its Alteration by Caloric Restriction in Mice J. Nutr., March 1, 2001; 131(3): 918S - 923. [Abstract] [Full Text]
|
|
|
|
|
|
S. X. Cao, J. M. Dhahbi, P. L. Mote, and S. R. Spindler Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice PNAS, September 11, 2001; 98(19): 10630 - 10635. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
